1. Field of the Invention
The present invention relates to compositions and methods for the detection, prognostic evaluation, and treatment of oncogenic disorders, particularly breast cancer.
Specifically, the instant invention provides for compositions, useful for identifying and treating disorders related to newly identified endogenous retroviruses which are present in a subset of humans, cats, and nonhuman primates.
2. Technical Problem Addressed by the Invention
Breast cancer (BC) is one of the leading causes of cancer death among women. The as induction of BC is thought to involve the interplay of several factors, including the genetic, hormonal, immunological and physiological status of the host, as well as dietary habits and exposures to chemicals, radiation or infectious agents. It is now clear that variations of several genes, including BRCA-1 and BRCA-2, can result in greatly increased risks for development of BC. However, defects in known BC susceptibility genes account for only about 5% of BC, the so-called familial cases (Armstrong et al., 2000; Gayther et al., 1998).
As with other types of cancer, the possibility that a virus is etiologically involved in sporadically occurring BC has not been eliminated. Consequently, there has long been a need to determine which, if any viruses are causally linked to the development of BC. The identification of such a virus would likely provide invaluable aid in the following areas BC medicine: prevention, diagnosis, determination of prognosis, and treatment.
3. Description of Related Art
Mouse mammary tumor virus (MMTV), a B-type retrovirus, was discovered during studies of hereditary cancer in mice at the Jackson Laboratories in the 1930""s (Bittner, 1936). As the prototype of slow-transforming retroviruses, MMTV has been definitively shown to cause BC in mice. Prior studies established that MMTV is transmitted both in the gerinline as endogenous proviruses and exogenously via milk. As endogenous elements MMTV proviruses follow patterns of Mendelian inheritance, as other sequences in the genome (Cohen et al., Cell,1979; Cohen and Varmus, 1979, 1980; Traina et al., 1981; Traina-Dorge and Cohen, 1983; Traina-Dorge el al., 1985; Varmus et al., 1978). Horizontal transmission of MMTV typically occurs by infection of mouse pups by MMTV virions present in the milk of infected dams. Thus, it is possible to transmit MMTV to mice by foster feeding. 30 or more unique proviral integration sites for endogenous MMTV have been identified. However, some wild mice do not carry any endogenous MMTV proviruses (Cohen and Varmus, 1979; Cohen et al, 1982). This result suggests that the many endogenous MMTV proviruses are relatively recent additions to the mouse genome. The most likely explanation is that MMTV entered the germline of certain mice (but not others) on multiple occasions after the evolutionary splits among the various species and subspecies of the genus Mus. Certain endogenous MMTV can be activated by hormones to form infectious virions capable of inducing mammary carcinomas after long latency periods. Most endogenous MMTV proviruses are defective and do not encode for infectious virions.
The MMTV Orf protein can function as a superantigen (SA). When expressed in the thymus during fetal/early development it can mediate complete or incomplete deletion of SA-reactive T-cells. SA expression is required to activate B-cells targets of MMTV in the gut-associated lymphoid tissue of nursing pups. Complete deletion of the SA responsive clones thus renders the mice resistant to MMTV infection in the gut and thereby leads to a low incidence of MMTV-induced tumors. On the other hand, in mice with only partially deleted responsive clones of lymphoid cells the SA activation stimulates expansion of the targets and spread of MMTV. As female infected animals reach puberty, estrogenic hormones drive expression of the MMTV long terminal repeat (LTR) through its hormone response element (HRE). This permits production and assembly of MMTV and spread of the virus to other hormonally-sensitive tissues, including the breast and ovaries. Integration of MMTV LTRs adjacent to certain cellular genes, such as the proto-oncogenes Int, Wnt and Fgf, can increase expression of these genes resulting in BC and other cancers.
The molecular genetic interactions between MMTV, the immune system of its murine host, and the breast and other hormonally-sensitive cells malignantly transformed by this retrovirus have been extensively studied. MMTV promotes mammary gland cancer in mice by insertional mutagenesis (Varmus et al., 1978; Varmus, 1985). MMTV proviral LTR elements direct steroid hormone-dependent transactivation of various cellular oncogenes including Wnt, Fgf and Int thereby promoting clonal expansion of tumor cells (Shackleford and Varmus, 1987, Shackleford et al, 1993; Jakobovits et al., 1986; Nusse, 1991; Nusse et al.,1985). For productive, persistent infection and completion of its replication cycle, MMTV must contain a superantigen and interact with a functional host immune system (Golovkina et al., 1995; Luther and Acha-Orbea, 1996; Coffin, 1992).
The discovery of the oncogenic MMTV has prompted many investigators to explore a retroviral etiology for BC in humans (Sarkar, 1980). Data collected over the past five decades has suggested the existence of a human homologue of MMTV. In 1971, Moore and associates reported that 60% of human milk samples from BC patients contain B-type particles indistinguishable from MMTV by electron microscopy, compared to 5% of the general population (Moore et al., 1971). These investigators also reported that 39% of Parsi women of India, an inbred population with a two-fold increased incidence of BC, had B-type particles in their milk (Das et al., 1972; Moore, 1971). Several studies have demonstrated that BC cells, but not cells from normal tissues, also contain reverse transcriptase (RT), an enzyme associated with all retroviruses. Numerous investigators have examined serum and breast milk for the presence of antibodies reactive with MMTV. Most of these studies were performed in the pre-AIDS era, prior to the advent of highly sensitive and specific techniques for detecting anti-retroviral antibodies made necessary for detection of HIV antibodies in donated blood.
Despite the numerous electron microscopic, biochemical and immunological studies on human breast carcinoma tissue, milk, patients"" sera, and breast carcinoma cell lines suggesting the existence of a human homologue of MMTV, proof that such an agent exists has remained elusive (Andersson et al., 1996; Ziegler, 1997). Most authors have dismissed the importance of prior studies purporting to show evidence of a human homologue of MMTV because of the presence of numerous human endogenous retroviruses (HERVs) (Larsson et al, 1994; Li et al., 1996; Lower et al., 1996; Meese et al., 1996; Ono, 1986; Patience et al., 1996; Faff et al., 1992). There are about 50,000 HERVs or HERV-related sequences in the human genome, some of which have been shown to have up to 60% homology to MMTV. In this regard, it is important to note that seroreactivity to HERV-K10, to this point the HERV considered to be most closely related to MMTV, cannot account for MMTV-reactive antibodies present in the sera of breast cancer patients and the smaller number of healthy individuals (Vogetseder et al., 1995). Furthermore, we believe that the presence of these MMTV-related sequences is precisely the reason that human homologues of MMTV have not previously been demonstrated conclusively by molecular techniques. The presence of these related, but distinct, sequences could have obscured the detection of more closely related sequences by prior investigators who used less sensitive techniques, such as Southern blotting.
Only recently have sequences with relatively high homology ( greater than 90%) to those of MMTV been isolated from human BC tissue (Wang et al., 1995, 1998;). Sequences 95-99% similar to MMTV env were amplified by PCR in 121 (38.5%) of 314 unselected breast cancer tumor samples. It is pertinent to note that the MMTV-like sequences were detected in only 2 (1.8%) of 107 breast specimens from reduction mammoplasties and in 0/80 samples from normal tissues or non-breast tumors. The MMTV-env like RNA was expressed (as determined by RT-PCR) in 66% of DNA PCR positive breast tumors (Wang et al, 1998). A complete 9.9 kb provirus with 94% similarity to MMTV was detected in 2 breast tumors. FISH (fluorescence in situ hybridization) revealed integration at several sites in DNA derived from BC tumors, but not normal breast cells (Wang et al., 1999 ACR mtg. abstracts #2933, 2944). Wang et al. suggested the existence of a human mammary tumor virus (HMTV) that is spread by the exogenous route of infection (horizontal transmission). Attempts by these and other investigators to amplify other regions of MMTV-related viruses, from the genomic DNA or cDNA of subjects who did not have BC, yielded HERV sequences (such as HERV-K10) with only about 60% homology to MMTV. Thus, BC tissues are the only tissues in which sequences that are highly similar to those of MMTV have been heretofore found. Consequently normal breast tissue and other tissues appeared to be negative for the expression of viruses with high homology to MMTV.
The retrovirus replication cycle is characterized by conversion of the single-stranded RNA viral genome into doubled-stranded proviral DNA by the multiple enzymatic activities of the virion-associated reverse transciptase (RT). As is the case with other retroviruses, integration of MMTV proviral DNA into the genome of host cells is required for expression of viral proteins and production of infectious progeny. In both infected mouse mammary glands as well as heterologous cells MMTV proviral DNA is integrated into a large number of apparently random sites. Integration of MMTV proviruses containing transcriptionally active LTR near some cellular genes (proto-oncogenes), such as Int, Wnt, and Fgf can result in over-expression of these genes, cellular transformation and clonal expansion of the tumor cells (Varmus, 1985; Shackleford et al., 1993; Jakobovits et al., 1986; Cohen, 1980; Breznik and Cohen, 1982). The long latency of MMTV-induce carcinogenesis is explained in part by the necessity for proviruses to integrate into these particular sites.
Genetic differences among viral strains of MMTV can account in part for the varying incidence of BC in diverse strains of mice. Mice of the C3H strain have a greater than 90% incidence of BC, compared to a  less than 1% incidence of BC in BALB/c mice. BALB/c mice foster-nursed on C3H females have a high incidence of BC which suggested that the tumorigenic MMTV of C3H can be horizontally transmitted in the milk (Bittner, 1936). Conversely, when C3H mice are foster nursed on a BALB/c female the incidence of BC is significantly lower (22-55%), but not as low as low-incidence mouse strains. This latter observation underscores the importance of the horizontally-transmitted milk-born virus in the high incidence strains, but also indicates the substantial differences in tumorigenic potential among endogenous MMTV proviruses. Proviruses of various mice with high and low tumor incidence could be distinguished by differences in solution hybridization kinetics and restriction endonuclease digestion patterns (Cohen et al Cell, 1979; Cohen and Varmus, 1979, 1980; Traina-Dorge and Cohen, 1983; Breznik et al. 1984). Using restriction enzymes that differentiate between hypomethylated and methylated DNA, proviruses of milk-borne MMTV were also shown to be hypomethylated, whereas most endogenous proviruses contain abundant 5-methylcytosine (Cohen, 1980; Breznik and Cohen, 1982; Breznik et al., 1984). Because hypomethylation is associated with increased gene expression, this observation could explain the importance of horizontally-transmined milk-borne MMTV in mouse strains with a high incidence of BC. Specific hypomethylation of an endogenous MMTV provirus was associated with expression of a 1.6 kb transcript of the lactating mammary gland of the BALB/c mouse (Traina-Dorge et al., 1985).
In other studies, endogenous MMTV proviruses have been shown to segregate as stable genetic units during inbreeding and that certain of these endogenous proviruses are transcriptionally active (Cohen et al., Cell, 1979; Cohen and Varmus, 1979, 1980; Traina et al., 1981; Traina-Dorge and Cohen, 1983; Traina-Dorge et al., 1985; Varmus et al., 1978). Recombinant inbred (RI) strains of mice have been used to define MMTV provirus composition and chromosomal locations (Traina et al., 1981; Traina-Dorge and Cohen, 1983). RI strains were developed by crossing mice from two highly inbred lines, randomly mating the F2 generation brothers and sisters and maintaining each as separate lines. Many genetic loci were mapped to specific chromosomes in these strains. By analyzing the segregation patterns of various MMTV proviruses with these genetic markers by restriction endonuclease analysis and Southern blotting, it was possible to determine the chromosomal locations of a number of MMTV proviruses in these strains and to establish that these provirus segregate by the rule of Mendelian inheritance. These and other analyses defined 10 distinct MMTV units (proviruses) in these animals some full length and some truncated. The ratios of inheritance for most of the MMTV proviruses were consistent with simple single-gene inheritance, though three of the MMTV units demonstrated some variance. Most noticeable was the presence of a unit contributed by one parent that was present in 23 of the 26 RI strains analyzed. Specific proviral units were identified that were transcriptionally active and associated with increased tumor production (Traina-Dorge et al., 1985). Importantly, cellular genes were identified that were significantly associated with virus expression showing the involvement of host genetics in disease progression (Traina-Dorge et al., 1985).
Several lines of evidence indicate that the endogenous MMTV proviruses have integrated relatively recently in the germline of various strains of mice (Cohen and Varmus, 1979; Varmus et al., 1978). If MMTV evolved from elements present in a progenitor of Mus musculus (the laboratory mouse), it would be expected that all individual mice would have similar MMTV proviruses. However, both laboratory mice and wild mice trapped at several locations having quite variable numbers and distributions of germline integration sites for their MMTV proviruses (Cohen and Varmus, 1979). The most striking finding among these results were the presence of some wild caught animals that contained no endogenous MMTV proviruses. The most likely interpretation of these results is that the endogenous MMTV proviruses arose by multiple independent integrations into the DNA of germinal cells after speciation of the genus Mus, rather than arising from genetic elements present in the evolutionary progenitors of mice.
The present invention provides for recombinant DNA molecules derived from one or more mammary tumor viruses which are endogenous retroviruses with homology to the sequences of MMTV. Specifically, the sequences of the instant invention have at least 99% identity with all or a portion of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 21, 23, 25, 27, or 29 or at least 92% identity with SEQ ID NO: 10.
Additionally, the present invention provides for the RNA molecules produced by is transcription of the DNA described above. Furthermore it provides for the polypeptides resulting from the in-frame translation of RNA which has been transcribed from the MTV DNA of the instant invention. According to the present invention, the referenced DNA sequences may be derived from any suitable source, such sources may include, but are not limited to, human, cat, and rhesus macaque.
The present invention also provides for a recombinant DNA plasmid (a vector) which comprises mammary tumor virus (MTV) DNA the sequences of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 10, 21, 23, 25, 27, or 29, or sequences which have at least 99% identity thereto. That is said DNA sequence is incorporated in the vector.
In one embodiment of the present invention the vectors further comprise a heterologous promoter operably linked to the MTV sequence (i.e. joined in the proper reading frame so as to be capable of producing functional MTV RNA and/or protein in vivo and in vitro). In one aspect of this embodiment of the invention the vector, which comprises the MTV DNA, is capable of episomal replication or chromosomal integration in at least one of the following cell types: bacterial cells, yeast cells, insect cells, avian, cells, and mammalian cells (this list of cell types is representative and should not be considered exhaustive). In another aspect, of this embodiment of the invention, the heterologous promoter provides for the expression of the MTV DNA sequence in one or more cell types. Cell types considered useful as part of this aspect of the invention include, but are not limited to the following: bacterial cells, yeast cells, insect cells, avian, cells, and mammalian cells.
According to another embodiment of the instant invention the MTV DNA sequences described above may be used to provide a method of detecting the presence of MTV DNA or RNA in a sample (of biologicalorigin, such as serum, or otherwise).
Another embodiment of the instant invention provides for a method of determining whether a sample contains antibodies which recognize proteins derived from the MTV DNA sequences described above (e.g. polypeptide sequences derived from transcription and translation of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 10, 21, 23, 25, 27, or 29). As a corollary to this aspect, the instant invention also provides for antibodies which specifically detect one or more of the polypeptides of the instant invention.
Another embodiment of the invention provides for diagnostic kits useful for detecting DNA, RNA, or polypeptides, from a mammary tumor virus, in a biological or other type of sample.
Another embodiment of the instant invention provides for methods of attenuating or eliminating the activity of MTV in its host animal. Various aspects of this embodiment provide for pharmaceutical compositions.,comprising substances which disrupt the activity of the MTV reverse transcriptase, protease, or integrase enzymes. Other aspects of this embodiment provide for pharmaceutical compositions capable of eliciting an immune response in a host animal.